A novel approach to exogenously manipulate the T cell antigen receptor (TCR) on T cells has been found that increases the sensitivity of these lymphocytes to identify and attack cancer cells in mice. This approach holds promise to function as an effective immunotherapy against metastatic cancers in humans using monovalent Fab fragments (Mono-Fabs) specific for the CD3 complex that is associated with the TCR. In the absence of strong antigen, anti-CD3 Mono-Fabs do not stimulate T cell immune function because they do not crosslink receptors for intracellular signaling. However, when exposed to antigen, T cells treated with anti-CD3 Mono- Fabs increase their level of response to weak antigenic stimulation in vitro, indicating that the Fabs bound to CD3 alter the TCR/CD3 complex and enhance T cell triggering, an effect we denominate ?receptor co- potentiation?. When tested in mice in vivo, anti-CD3 Mono-Fabs display therapeutic properties against metastatic melanoma that depend on the presence of T cells able to recognize antigens associated with the tumor. Since anti-CD3 Mono-Fabs target the TCR/CD3 complex, testing combinations with alternative immunotherapies that target other receptors on T cells was. Anti-CD3 Mono-Fabs showed a synergistic effect with the alternative immunotherapies, including checkpoint inhibition or adoptive cell therapy, which resulted in significantly extended survival of the mice tested. Now, Mono-Fabs specific for human CD3? have been identified that may mediate T cell co-potentiation in vitro, as well produce anti-tumor T cell responses in two different and ccomplementary humanized-mouse models of metastatic melanoma. In the present proposal, transgenic mice expressing human CD3? will be crossed with inbred and outbred mouse strains in order to produce different degrees of genetic diversity to resemble adult humans. Additionally, so called ?dirty mice? from pet stores will also be co-housed with human CD3?-transgenic mice to induce levels of ?foreign? and immune experience that better correspond with those observed in adult human immunotherapy patients. Mice from these various conditions will be used to determine the extent to which candidate anti-human CD3? Fabs display anti-tumor potential against metastatic melanoma (aim 1), in single and combination immunotherapies (with checkpoint inhibition and adoptive cell theapy), as well as the extent to which immunotoxicity occurs (aim 2). Results will inform the choice of optimal candidate(s) to subsequently undergo development for anti-cancer translational application. Finally, the CD3 signaling mechanism promoted by anti-human CD3e Fabs will be studied in a mechanismtic manner to better understand T cell co-potentiation (aim 3). If successful, this project will produce a novel modality of immunotherapy that may be exploited alone and/or to synergize other immunotherapies, and will provide a biochemical mechanistic basis for this function. The hope is that soon thereafter, experimentation might advance these studies to translational application with an ultimate goal of helping prolong survival of patients suffering from metastatic melanoma and additional malignancies.